In order to analyze the genetic regulation of T cell responses to NASE, a series of cloned lines were generated in BALB/c (H-2d) as well as (H-2b x H-2a)F1 T cells. Individual clones were restricted to recognizing NASE in the context of either A(alpha)A(beta) or E(alpha)E(beta) products. The antigen fine specificity of cloned NASE-specific T cells was also probed through the use of mutant NASE molecules and synthetic peptides corresponding to segments of NASE. A consistent correlation was found between the fine specificity of a given clone and its MHC restriction specificity. A(b)(alpha)A(b)(beta) restricted clones were selectively responsive to peptide 91-110; E(k)(alpha)E(k)(beta) restricted clones were responsive to peptide 81-100. The nature of the immunogenic peptides which are processed and presented to T cells was further evaluated using a panel of variant NASE peptides. It was observed that negative (inhibitory) interactions appear to occur between amino acids in peptides which interfere with T cell responses. Single amino acid changes, by eliminating such apparent negative interactions result in the restoration of T cell stimulatory ability in these peptides.